1. Field of the Invention
This invention relates to a method of solid phase immunoassay incorporating a luminescent label for the quantitation of antigen, hapten or antibody analyte in a liquid sample or analyte occuring on or attached to cells or other particulate material.
2. Description of the Prior Art
A number of methods exist for the detection of substances of biological origin. One large class of methodology is the immunoassay, where antigens (or haptens) and their corresponding antibodies are used to probe the sample for each other. One very important variant of the immunoassay is the solid phase immunoassay. (Cf. Catt et al., J. BIOCHEM, 100: 31c (1966); Catt et al., SCIENCE, 158: 1570 (1967); U.S. Pat. No. 3,646,346 by Catt et al., these references and patents, and subsequently cited references and patents in the context in which they appear, are incorporated by reference thereto).
Radioactive atoms, siuch as .sup.125 I, .sup.131 I, .sup.3 H, and .sup.14 C for example, are commonly utilized as the label in solid phase immunoassays. The resulting solid phase radioimmunoassays are quite sensitive but suffer commonly recognized disadvantages. The radioactive nature of the label subjects the assay to stringent regulatory requirements, results in a relatively short reagent shelf life and poses a waste disposal problem.
In an attempt to overcome the disadvantages of radioimmunoassays, several alternative labeling methods have been developed. Foremost among these are the enzyme immunoassays (EIA, ELISA) where an enzyme replaces the radioactive label. (cf. U.S. Pat. No. 3,551,555 by Schuurs). Enzymes commonly utilized as labels are horseradish peroxidase, alkaline phosphatase, B-galactosidase and glucose oxidase. Enzyme immunoassays have an advantage over radioimmunoassays in that the enzyme labels are very stable and special facilities and instrumentation are not required. However, enzyme immunoassays are generally slower and more tedious to perform than radioimmunoassays.
Luminescent labels have been utilized as an alternative to radioactive or enzyme labels. (cf. U.S. Pat. No. 4,201,763 by Mothony et al.; U.S. Pat. No. 3,992,631 by Harte; U.S. Pat. No. 3,999,948 by Deindoerfer et al.; A. Coons, FLUORESCENT ANTIBODY METHODS; J. Danielli (Editor), GENERAL CYTOCHEMICAL METHODS Vol. 1). Fluorescein is the most commonly used label. Although fluorescence immunoassays possess the ease of use advantage of radioimmunoassays and the reagent stability advantage of enzyme immunoassays, prior art fluorescence immunoassays lack the sensitivity of either radioimmunoassays or enzyme immunoassays. This lack of sensitivity has significance in both research and clinical applications with the result that fluorescence immunoassays have seldom been the assay of choice in these applications.